响应面试验优化广昌白莲蛋白提取工艺及其酶解多肽抗氧化活性

以广昌白莲为原料，通过单因素试验和响应面试验优化超声波辅助提取白莲蛋白的工艺，并采用胃蛋白酶和碱性蛋白酶对白莲蛋白进行单酶和有次序双酶水解，研究其酶解产物的抗氧化活性。结果表明，广昌白莲蛋白的最佳提取条件为：在超声功率固定250 W条件下，料液比1∶14（g/mL）、提取温度35.5 ℃、提取时间58 min、NaCl浓度0.14 mol/L。所得蛋白提取率达到89.86%，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfatepolyacrylamidegelelectrophoresis，SDS-PAGE）分析显示其分子质量主要分布在14～25 kD和35～60 kD之间；不同酶解方式显著影响了白莲蛋白的酶解效果及其酶解产物的抗氧化活性，其中先胃蛋白酶后碱性蛋白酶的双酶酶解为最佳方式，其蛋白水解度达到20%，其酶解产物（P-A）H的总还原能力（A700 nm）、1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2- picrylhydrazyl，DPPH）自由基清除能力和·OH清除能力IC50值分别达到1.002、0.379 mg/mL和1.093 mg/mL，表明广昌白莲蛋白酶解产物具有较强的抗氧化活性。

Optimization of Extraction Conditions of Guangchang White Lotus Seed Protein by Response Surface Methodology and Antioxidant Activities of Its Enzymatic Hydrolysates

The conditions for ultrasonic-assisted extraction of protein from Guangchang while lotus seed were optimized
by single factor experiments and response surface methodology. The extracted proteins were hydrolyzed using alcalase and
pepsi singly or in sequential combinations, and antioxidant activities of the resulting hydrolysates were analyzed. The results
suggested that the optimum conditions under a fixed ultrasonic power of 250 W for extracting protein from Guangchang
white lotus seed were determined as follows: solid/solvent ratio, 1:14; extraction temperature, 35.5 ℃; extraction time,
58 min; and NaCl concentration, 0.14 mol/L. Under these conditions, the extraction yield of protein was 89.86%. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that molecular weights of the extracted
proteins were mainly in the range of 14–25 kD and 35–60 kD. Different treatments with pepsin and/or alcalase significantly
influenced the effectiveness of enzymatic hydrolysis of lotus seed protein and antioxidant activities of hydrolysates, and
sequential treatment with pepsin followed by alcalase was the best one. Under this treatment, the degree of hydrolysis
was 20%, and the total reduce power (A700 nm), 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50)
and hydroxyl radical scavenging activity (IC50) of the hydrolysate (P-A)H were 1.002, 0.379 mg/mL and 1.093 mg/mL,
respectively, demonstrating that the enzymatic hydrolysate had potent antioxidant activity.